CN101061385A - A biological marker for inflammation - Google Patents
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- CN101061385A CN101061385A CNA2005800391767A CN200580039176A CN101061385A CN 101061385 A CN101061385 A CN 101061385A CN A2005800391767 A CNA2005800391767 A CN A2005800391767A CN 200580039176 A CN200580039176 A CN 200580039176A CN 101061385 A CN101061385 A CN 101061385A
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Inflammatory state in a subject is assayed by determining the level of expression of A3 adenosine receptor (A3AR) in white blood cells (WBC), e.g. circulating WBCs, from the subject. A high level of expression of A3AR is indicative of an inflammatory state in the subject. This assay can be used for determining the severity of inflammation in a subject and monitoring the efficacy of anti-inflammatory treatment. Also, the level of expression may be used for selecting patients to receive an anti-inflammatory treatment that comprises an A3AR agonist.
Description
Invention field
The present invention is in the effect of diagnosis inflammation and definite inflammation treatment, use therefore particularly that the biological marker relevant with inflammatory conditions carry out the field.
Prior art
Be that row are considered to the state of the art relevant prior art interior with describing the field of the invention below.The quoting of these references (Acknowledgement) represented by indicating to number from its of tabulating below in bracket in this manual sometimes.
1.?Fishman?P，Madi?L5?Bar-Yehuda?S，Barer?F，Del?Valle?L，Khalili?K.Evidence?for?involvement?of?Wnt?signaling?pathwayin?IB-MECA?mediated?suppression?of?melanoma?cells.Oncogene.，
21：4060-4064(2002).
2.?Fishman?P，Bar-Yehuda?S5?Rath-Wolfson?L5?Ardon?E5?BarrerF5?Ochaion?A5?Madi?L.Targeting?the?A3?adenosine?receptor?forcancer?therapy：inhibition?of?Prostate?carcinoma?cell?growthby?A3AR?agonist.Anticancer?Res.，
23：2077-2083(2003).
3.?Madi?L5?Bar-Yehuda?S，Barer?F5?Ardon?E5?Ochaion?A5Fishman?P.A3?adenosine?receptor?activation?in?melanoma?cells：association?between?receptor?fate?and?tumor?growth?inhibition.J.Bio.Chem.，
278：42121-42130(2003).
4.?Ohana?G5?Bar-Yehuda?S5?Arich?A5?Madi?L5?Dreznick?Z5Silberman?D5?Slosman?G5?Volfsson-Rath?L，Fishman?P.Inhibitionof?primary?colon?carcinoma?growth?and?liver?metastasis?by?theA3?adenosine?receptor?agonist?IB-MECA.British?J.Cancer.，
89：1552-1558(2003).
5.?Fishman?P，Bar-Yehuda?S，Ohana?G，Ochaion?A，EngelbergA，Barer?F，Madi?L.An?agonist?to?the?A3?adenosine?receptorinhibits?colon?carcinoma?growth?in?mice?via?modulation?of?GSK-3β?and?NF-κB.?Oncogene，
23：2465-2471(2004).
6. U.S. Patent application 2004016709 A1.
7. Szabo, C, Deng people Suppression of macrophage inflammatoryprotein (MIP)-1 α production and collagen-induced arthritisby adenosine receptor agonists. British J.Pharmacology
125: 379-387 (1998).
8. Mabley, J., wait people The adenosine A3 receptor agonist, N6-(3-iodobenzyl)-adenosine-5 '-N-methyluronamide, isprotective in two murine models of colitis.Europ.J.Pharmacology
466: 323-329 (2003).
9. Baharav, E. waits people The effect of adenosine and the A3adenosine receptor agonist IB-MECA on joint inflammation andautoimmune diseases models.Inter.J.MoI.Med.
10(supplement1) page S 104, and abstract 499 (2002).
10. PCT application, publication number WO2005/0063246, title are " Method forTreatment of Multiple Sclerosis ".
11. Montesinos, M.Carmen waits people Adenosine A
2AOr A
3Receptorsare required for inhibition of inflammation by methotrexate andits analog MX-68.Arthritis ﹠amp; Rheumatism,
48: 240-247 (2003).
12.?Madi?L，Ochaion?A，Rath-Wolfson?L，Bar-Yehuda?S，Erlanger?A，Ohana?G，Harish?A，Merimski?O，Barer?F，Fishman?P.The?A3?Adenosine?Receptor?is?Highly?Expressed?in?Tumor?vs.Normal?Cells：Potential?Target?for?Tumor?Growth?Inhibition.Clinical?Cancer?Research，
10：4472-4479，2004.
13. U.S. Patent application, publication number 20040137477 A1, title are " A3AR as amarker for a diseased state ".
14. Gessi, people Elevated expression of A such as S.
3Adenosinereceptors in human colorectal cancer is reflected in peripheralblood cells Clinical Cancer Research
10: 5895-5901,2004.
Background of invention
A
3Adenosine receptor, G
iAlbumen correlativity cell surface receptor is considered to anticancer and target anti-inflammatory.This receptor is highly expressed in various tumor cell types, but relatively low at adjacent normal tissue expression.Activate this receptor by the synthetic activator of specificity and induce the adjusting of the downstream signal transduction pathway that comprises Wnt and NF-kB, thereby cause the inhibition (1-5) of tumor growth.
Research has shown A in the body
3The AR activator suppresses the development of colon cancer, prostate cancer and cancer of pancreas and melanoma and liver cancer.Also show A
3The AR activator is brought into play the effect of antiinflammatory by improving inflammatory processes in different experimental autoimmunity model (for example rheumatoid arthritis, Crohn disease and multiple sclerosis (6-10)).A has also been proposed
2AAnd A
3The antiinflammatory action of receptor-mediated methotrexate (11).
Compare A with normal cell
3Adenosine receptor (A
3AR) expression is enhanced (12) in cancer cell.Therefore, with A
3The expression of AR is described as being used for the method (13) of cancer diagnosis.In addition, A has also been described
3The expression of AR is enhanced (14) in suffering from the peripheral blood of patients cell of colorectal cancer.
General description of the present invention
The purpose of this invention is to provide the method that is used for determining inflammatory conditions the experimenter.
Another object of the present invention provides the method that is used for determining the experimenter severity of inflammatory conditions.
Another object of the present invention provides the method for the effect of the anti-inflammatory treatment processing that is used for definite experimenter.
A further object of the present invention provides and is used to select the experimenter to accept the method for anti-inflammatory treatment processing.
The present invention is based on astonishing discovery, promptly compare A in the WBC of the experimenter with inflammatory conditions with the experimenter of health
3The adenosine receptor expression levels increases.In addition, find in the experimenter that drug therapy reacts to anti-inflammatory the A among its WBC
3The expression of adenosine receptor reduces.This is found to be A
3The expression of adenosine receptor has been paved road as the method for other application of diagnosing inflammatory conditions and describing below.
In a first aspect of the present invention, the method for inflammatory conditions among definite experimenter is provided, this method comprises to be determined from experimenter's leucocyte (WBC) A in the circulating leukocyte for example
3Adenosine receptor (A
3AR) expression.A
3The high level expression of AR shows have inflammatory conditions in the experimenter.
The sample that comprises WBC can be that whole blood maybe can be the blood constituent that comprises WBC.Sometimes, may want to use and comprise for example monocytic subpopulation of special group, MNC-of mononuclearcell (MNC) or lymphocyte or the lymphocytic subpopulation composition of T cell, B cell or its subpopulation for example of WBC.Sometimes also can for example obtain to comprise the sample of WBC from lymphatic system from lymph node.
Can be by determining A
3The level of AR mRNA and/or A
3The level of AR albumen is determined expression levels.Therefore term " expression " used herein comprises the A in the cell of sampling
3The level of AR mRNA and A
3AR albumen or A
3The level of AR protein fragments.
Find that medicinal treatment can influence A
3The AR expression levels.Therefore, the medical history in past comprises before the treatment or the medical history during the treatment, can influence A
3The expression of AR, thus in carrying out method of the present invention, may need it is taken into account.
In a second aspect of the present invention, the method that is used for determining the experimenter severity of inflammatory conditions is provided, it comprises determines A among the experimenter WBC
3The AR expression levels; With with A in the cell
3The expression of AR and the level of predetermined standard compare, and the level of described predetermined standard makes A
3The expression of AR is related with the severity of infection.Described predetermined standard can comprise, for example makes the related value in groups of measurement of the severity of result and inflammation, and it can be numerical tabular or the continuous curve that disperses; Or it can be a descriptor in groups, for example about the possible outcome of inflammation severity or the qualitative tabulation of its meaning, if during for example with the color reaction performance results, described descriptor can be listed the possible color of relevant inflammation severity or scope and its meaning of color intensity result; Or it can comprise the chart or the caption of the expection measurement result of the different severities of inflammation or different inflammatory conditions; Or the reference standard that can produce together with the sample that is used for data calibration and assessment in groups.Usually can be by measuring A in a plurality of samples (it is the sample from each state in many inflammatory disease states)
3The expression of AR is to obtain obtaining described standard about the statistical measurement of the association between expression and the morbid state.The classification of morbid state can be a binary for example: light inflammation and serious inflammation.Described classification also can comprise multiple different state, for example, and slight, moderate and serious inflammation.Also can be by according to expression levels, use numerical example such as correspondence on the lenient side to heavy inflammation 1 and 10 between numeral etc. classify.As will be recognized, may there be the classification of many types and the classification that the present invention is not limited to particular type.
In a third aspect of the present invention, the method for the effect of the anti-inflammatory treatment sex therapy that is used for definite experimenter is provided, this therapy comprises to the experimenter uses A
3The AR activator.This therapy can be to use A
3The monotherapy of AR or A
3AR and another kind of medicine be A for example
3The conjoint therapy of AR and methotrexate combination.This method is included on two or more continuous time points to be determined from the A among experimenter's the WBC
3The expression of AR, at least one described time point is during anti-inflammatory treatment, and wherein the difference on the level is represented the effect of this drug therapy.Described continuous time point can be one or more time points and the one or more time points of treatment time phase that for example adopt before the anti-inflammatory treatment, the one or more time points that adopt between the one or more time points that adopt during the treatment and treatment stand-down.
According to embodiments more of the present invention, the A among the WBC
3The AR expression can be used for determining the state or the severity of inflammation, for example is used for determining the existence of inflammatory conditions or not existing.According to a further embodiment of the present invention, A
3The expression of AR can be used for the quantitative of severity degree of inflammatory conditions and determines.Therefore, the term that herein uses " is determined " or " decision " comprises quantitatively and qualitative definite.
" inflammatory conditions " comprises any state active or subclinical inflammation.According to the preferred embodiment of the invention, the present invention is used for determining the experimenter who suffers disease and autoimmunity inflammatory disease the state of inflammation.This inflammation can cause owing to inflammatory disease, or it can be the spinoff of the disease or the illness of some other types.The example of inflammatory disease includes but not limited to: inflammatory bowel disease, inflammatory cell, fibrous inflammatory hyperplasia, inflammatory gallbladder disease, inflammatory papillary hyperplasia and autoimmune disease.Autoimmune disease can comprise any following disease: rheumatoid arthritis, myasthenia gravis (MG), CMG, multiple sclerosis (MS), stiff man syndrome (Stiff-man syndrome), torrid zone spastic paralysi, the RasmussenShi encephalitis, acute motor axonal neuropathy, acute sensation-motion axonal neuropathy, the dorsal root ganglion neuritis, acute whole plant nerve DPN, BN, acute bleeding gangrenosum acne leukoencephalitis (Acute necrotizing hemorrhagiclekoencephalitis), sporadic gangrenosum acne myelopathy, the paraneoplastic cerebellar degeneration, guillain-Barre syndrome, limbic encephalitis, opsoclonus-myoclonia incoordination (Opsoclonus-myoclonus ataxia), the sensory neuron inflammation, AN, demyelinating neuropathy, the dull-witted complex of AIDS-, tourette (family name) syndrome, Miller-Fisher syndrome, Alzheimer (family name) disease, Robert Graves (family name) disease, this (family name) thyroiditis of bridge, postpartum thyroiditis, focal thyroiditis, teenager's thyroiditis, idiopathic hypothyroidism, I type (insulin-dependent) diabetes, A Disen (family name) disease, hypophysitis, autoimmunity urine collapses, hypoparathyroidism, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid (Bullous phemphigoid), pemphigoid gestationis, scar class pemphigus, dermatitis herpetiformis, acquired epidermolysis bullosa (epidermal bullosaacquisita), erythema multiforme, herpes gestationis, leucoderma, chronic urticaria, discoid lupus, general alopecia/alopecia areata, psoriasis, oneself immunity hepatitis, PBC, chronic active hepatitis, chronic active hepatitis/PBC overlap syndrome, primary sclerotic cholangitis, autoimmune hemolytic anemia, ITP, Evans syndrome, the thrombopenia that heparin brings out, the primary autoimmune neutropenia, baby's autoimmunity (primary) neutrophilic granulocytopenia, autoimmune neutropenia after the bone-marrow transplantation, acquired autoimmunity hemophilia (Acquired autoimmune hemophilia), autoimmunity gastritis and pernicious anaemia, CD, Crohn disease, ulcerative colitis, sialadenitis, the autoimmunity ovarian failure,premature, azoospermia, hypogonadism, the male sterility relevant with the sperm autoantibody, autoimmunity orchitis, ovarian failure,premature, AO, uveitis, the retinitis, sympathetic ophthalmia, Birdshot retinochoroidopathy, the Vogt-Koyanagi-Harada granulomatous uveitis, retinosis, lens-induced uveitis, optic neuritis, the autoimmunity sensorineural hearing loss, plum Neil (family name) disease, autoimmune myocarditis, CHB (neonatal lupus), just add this (family name) disease, adriamycin cardiotoxin (Adriamycin cardiotoxicity), DressierShi myocarditis is in conjunction with levying, bronchial astehma, between matter stringiness tuberculosis (Interstitialfibrosing lung disease), rapidly progressive glomerulonephritis, the autoimmunity tubulointerstitial nephritis, systemic lupus erythematosus (SLE), antiphospholipid syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, the expense ear is put forward (family name) syndrome, large granular lymphocyte increases (Large granular lymphocytosis) (LGL), Si Yegelun (family name) syndrome, Sjogren's syndrome disease (chorionitis), CREST syndrome, mixed connective tissue disease, polymyositis/dermatomyositis, Gourde(G) Paasche thorough (family name) disease, Wegener (family name) granulomatosis, churg-Strauss syndrome, purpura,Henoch-Schonlein, microcosmic polyangitis (Microscopic polyangiatis), periarteritis nodosa, black peculiar (family name) syndrome, atherosclerotic, one property crossed (huge) cell arteritis inflammation, aorto-arteritis, kawasaki disease, ankylosing spondylitis, Lai Te (family name) disease, Sneddonsdisease, autoimmune polyendocrinopathy, candidiasis-ectodermaldystropy, basic cryoglobulinemia vasculitis (Essentialcryoglobulinemic vasculitis), epidermis leukocytoclastic angiitis (Cutaneous leukocytoclastic angiitis), lime (family name) disease, rheumatic fever and heart disease, the EF, paroxysmal cold hemoglobinuria, the rheumatic polymyopathy, fibromyalgia, POEMS syndrome (polyneuropathy, the organomegaly, endocrine disease, M-spot and skin change), relapsing polychondritis, from centre immunity lymphoproliferative syndrome, TINU syndrome (acute tubulointerstitial nephritis and uveitis), common variable immunodeficiency, TAP (carrier relevant) defective with antigen presentation, Europe door Cotard (Omenn syndrome), high IgM syndrome, BTK agammaglobulinemia (disease), after human immunodeficiency virus and the bone-marrow transplantation.
The sample that comprises WBC that is used for the inventive method can comprise the cell of arbitrary known type of forming this colony.Especially, described sample should preferably include mononuclearcell (monocyte and/or lymphocyte).Sometimes sample can be extraly or is selectively comprised granulocyte (neutrophil cell, acidophic cell or basophilic granulocyte).
In the first embodiment, A
3The high level expression of AR is as the index of inflammatory conditions among the experimenter.Term " high level " is interpreted as that expression is significantly higher than the expression in the normal cell.For example, can be with A among the WBC
3The AR expression levels is compared with control level, and described control level is the A among health volunteer's the normal WBC
3The expression of AR.Sometimes, determine that by detection and the corresponding detected sample from individuality of one or more reference standard expression is useful, described reference standard is another reference standard of for example representing a reference standard of normal condition and representing inflammatory conditions; Or the reference standard of the reference standard of an expression normal condition and two or more expression various disease states.
In second embodiment, expression and the standard of determining compared.Described standard can based on the front determine from healthy individual with from having inflammatory conditions or having the level of the individuality of different inflammatory conditions.The form of discrete values for example, or be that the form with the chart of different colours with the healthy and inflammatory conditions of expression or tone provides described standard under the situation of colourimetry in determination method; Or the form of the comparison curves that can produce based on these standards provides.
Can be by determining to be present in the A in the WBC cell
3(as mentioned above, it can be A to the AR expression levels
3The level of AR albumen, protein fragments or mRNA level etc.) formulate these standards, described WBC cell available from a plurality of by diagnosis (by other modes, for example by the doctor, by histological techniques etc.) patient positive, that have the inflammation of different severity levels.Method that also can be by various routines for example is identified for formulating the severity of the disease of described standard by pathological technique.In another embodiment, simultaneously the experimenter's of many health volunteers and different inflammatory conditions standard is measured, the level that will determine in determined sample and these standards are relatively then.
For example, the protein content level between per 1,000,000 cell X1 to X2 can be defined as the index of 1 grade of inflammation, and the higher protein content between per 1,000,000 cell Y1 to Y2 may be defined as the index of 2 grades of inflammation etc.After these standards of formulation, just may be with A available from particular individual
3The value of the standard of AR expression levels and correspondence compares, thereby obtains the assessment of disease severity.
Can by before treatment, during the treatment and the sample of gathering WBC of the different time points after the treatment assess the effect of experimenter's anti-inflammatory treatment.For example, can gather first sample and can gather second sample by the time point during treating at the time point before the treatment beginning.Compare A in second sample with first sample
3The minimizing of AR expression represents that treatment is effective.The degree that reduces can be represented the degree of therapeutic efficiency, and promptly correlativity can be quantitative.
In another example, can gather first sample and can during treating, gather second sample on the time point after the time point of first sample on the time point during treating.Compare A in second sample with first sample
3The minimizing of AR expression represents that treatment is effective.
In the 3rd example, gather first sample on can the time point during treating and can on the time point behind the TD, gather second sample.In this case, compare A in second sample with first sample
3The increase of AR expression represents that treatment is effective.
Certainly, also can carry out other different combinations and surpassing collected specimens on 2 time points.
The present invention also provides the experimenter who is used to select to suffer certain inflammation disease to accept the method for anti-inflammatory therapy, and this anti-inflammatory therapy comprises to the experimenter uses A
3Adenosine receptor (A
3AR) activator, this method comprise the A among the WBC that determines the experimenter
3If the expression of AR and described level are higher than predetermined level, select this experimenter to accept described anti-inflammatory therapy.Certain threshold level that described predetermined level can be all experimenters.Described predetermined level also can be many levels of different patient colony, for example: the level of all ages and classes colony; The level of various disease state; (for example, the discovery methotrexate is induced A to the history of the level-past drug therapy of different medical histories
3The level of AR increases) ill year number etc.Can determine described predetermined level by clinical research, ACR20, an ACR50 that described research is for example formulated by American College of Rheumatology according to the acceptable response standard and a standard among the ACR70 or any other acceptable effective standard are sought the correlativity between expression of receptor and the drug response.
The selection that is suitable for the experimenter of anti-inflammatory therapy can be by the A in the WBC sample that picks up from described experimenter before definite treatment
3The expression of AR carries out.If the A that determines
3The level of AR is higher than predefined threshold value and then selects this experimenter.
According to an embodiment, threshold value is the certain multiple (certain multiple) of A3AR expression among health volunteer's the WBC.According to another embodiment, determine threshold value based on the average expression among the patient who suffers from described certain inflammatory disease, and it can be described mean value or certain multiple or its part.According to other embodiments, determine threshold value among the people patient based on the clinical research among the people patient, described research determines that through being designed for expression and patient are to the correlativity between the reaction of described cure.As being realized, can be different for different inflammatory disease threshold values.As also being realized, according to its character, this choice criteria is based on statistics, thereby the patient that expression is selected can be to treating aitiogenic a certain probability.Therefore, such choice criteria, as undoubtedly by understood by one of ordinary skill in the art, these choice criteria may not be made a prediction to reaction fully, and therefore the patient of some part of selecting thus also can be to treating nullvalent patient.
This system of selection also can be used for selecting to participate in the candidate of clinical research with the effect of detection anti-inflammatory therapy, and described therapy comprises A
3The AR activator individually or and other drug for example methotrexate use to the patient together.As recognized by those skilled in the art, clinical research (being also referred to as term ' clinical testing ' or ' clinical protocol ') is the definite new drug that carries out in people volunteer or the scientific research of the mechanism of action of cure in people experimenter.The new way of determining experimental therapy or using known therapies is tested in intervention, and whether safety is with effective under controlled environment.By clinical research, the doctor finds to prevent, detect, diagnose, control and treat the new and better method of disease.According to the present invention, selecting patient's clinical research based on the A3AR level can be the clinical research of I phase, II phase, III phase, IV phase or any other type.
Summary of drawings
In order to understand the present invention and to understand how to carry out it in practice, by indefiniteness embodiment, describe embodiment preferred with reference to the accompanying drawings now, wherein:
Figure 1A-1D describes and shows A
3The back exemplary Western blotting that quilt is raised in inflammation tissue and hematopoietic tissue and the average trace intensity of correspondence and the histogram of standard error take place in inflammation in AR.
Fig. 2 describes expression Western blotting and the average trace intensity of correspondence and the histogram of standard error relevant with the disease clinical score that is presented at A3AR in the AIA model." 0 " expression animal (animal of NIP) of not test (N.T.) and " 6 ", " 9 " and " 12 " relate to the animal and the inflammation in these animals of this numeral of inflammation and mark.
Fig. 3 shows control-animal and with the combination of methotrexate (MTX), CF101 (clinical grade IB-MECA), MTX and CF101 or only use the variation as the arthritis severity of the function of time in the AIA animal of carrier (contrast) processing.
Fig. 4 A-4B has described exemplary Western blotting and has had the histogram of standard error accordingly, and this figure shows lymph-node cell (Fig. 4 A) in the AIA animal after the animal, AIA animal of not test (N.T.) and CF101 (clinical grade IB-MECA) handle and the A in spleen (Fig. 4 B) cell
3The AR protein expression level.
Fig. 5 A-5C has described exemplary Western blotting and the corresponding histogram with standard error, and this figure shows with the A in pawl (Fig. 5 A), synovial tissue (Fig. 5 B) and the PERIPHERAL BLOOD MONONUCLEAR CELL (Fig. 5 C) among AIA vehicle treated or that handle with CF101
3The expression of AR albumen.
Fig. 6 has described Western blotting and corresponding histogram, the figure illustrates the animal, AIA animal of not test (N.T.) and the lymph node of the AIA animal of handling with MTX in A
3The expression of AR albumen.
Fig. 7 has described the A that shows among 7 health volunteers and 7 the RA experimenters
3The Western blotting of the expression of AR albumen and corresponding histogram.
Fig. 8 shows with CF101 3 months before and after treatment the A in the RA peripheral blood of patients mononuclearcell
3The histogram of AR level.For each patient, represent reaction with number percent according to ACR standard (formulate by American College of Rheumatology, determine the standard of effect of the medicine of treatment RA).
Fig. 9 shows among 4 patients that measure with arbitrary unit during (the black post) and treatment back (grey post) and treatment before the methotrexate for treatment A among the PBMNC
3The histogram of the expression intensity of Western blotting that AR expresses and correspondence.
Embodiment
Material and method
Inducing of the arthritis that adjuvant is induced in the rat (AIA) model
(Jerusalem Israel) obtains the 8-12 female Lewis rat in age in week from the Harlan laboratory.Provide tap water with the granulating food raising rat of calibrationization and for it.According to Can-Fite BioPharma, Petah Tikva, the guide that the management of laboratory animal council of Israel (Institutional Animal Care and Use Committee) formulates experimentizes.Give rat skin lower injection 100 μ l Mycobacterium tuberculosis (Mt) H37Ra (Difco, Detroit, USA) suspending liquid of Zu Chenging at the base portion of tail by incomplete Freund's adjuvant (IFA) and 10mg/ml heat-killed.Each group comprises 10 animals.
The 14th day begins to handle with IB-MECA (10 μ g/kg) after inoculation, Orally administered by gavage, handles for twice every day.Beginning in the 14th day after inoculation (1.5mg/kg) was once handled another group by using in the peritonaeum in per 3 days with methotrexate (MTX).Control group in each experiment is only accepted carrier (with corresponding to the DMSO in the dilution of drug dilution degree).
Following assessment clinical disease mobility scoring (Clinical Disease ActivityScore): the clinical arthritis of per two days inspection animals.Points-scoring system presents 0 to 4 fen to each limbs to be changed in the scope: 0-does not have arthritis; Rubescent or the swelling of a toe/articulations digitorum manus of 1-; 2-surpasses the rubescent and swelling of a toe/articulations digitorum manus; 3-ankle and shank-metatarsal joints are got involved; Rubescent or the swelling of the whole pawls of 4-.By scoring addition calculation clinical score with the leg of 4 individualities.Also according to (Japan) inflammation intensity is determined in the increase of the diameter of the rat hind paw of Ce Lianging for Mitotoyo, Tokyo by caliper.
Separating and the preparation of protein extract of inflammation tissue and hematopoietic tissue
A. inflammation tissue
Above ankle-joint, cut rear solid end.Bone tissue is cut into pieces, in liquid nitrogen snap frozen and-80 ℃ of storages until use.Be the preparation protein extract, in pawl tissue (4ml/gr tissue), add RIPA damping fluid (containing 150mM NaCl, 50mM Tris, 1%NP40,0.5% dexycholate and 0.1% SDS).With polyelectron that potpourri is also centrifugal in homogenate on ice.
Take out synovial tissue, by at 37 ℃ down under the strong concussion (200rpm), this is organized in incubation separated the synovial cell in 30 minutes among the RPMI that contains 1mg/ml clostridiopetidase A IV and 0.1mg/ml DNA enzyme.Collection contains synovial cell's supernatant and extracts indigested tissue again.To mix and use the PBS washed cell from the supernatant of extracted twice.The preparation protein extract.
B. hematopoietic tissue
Take out lymph node, at first then its pin by 22G is decomposed to come isolated cell by shredding this tissue.Take out spleen and it accepted LSM that (Nycomed AS, Oslo is Norway) to carry out the separation of mononuclearcell.The preparation protein extract.
From separating of RA patient and health volunteer's peripheral blood lymphocytes
Extract blood from health volunteer or RA patient.Use Ficoll-Hypaque gradient separations mononuclearcell (lymphocyte and monocyte).Extract albumen from mononuclearcell.
Clinical research
Extract blood from the RA patient who participates in the clinical research of subsidizing by Can-Fite BioPharma, in described research, estimate the effect of CF101 (clinical grade IB-MECA) the arthritic.The patient accepts 0.1,1.0 or 4.0mg CF101 randomly, twice of every day.Extract blood at two time points: (a) at the removing after date in 4 to 6 weeks after the previous treatment with before CF101 treatment beginning-this during be taken as baseline values; (b) treating with CF101 back 3 months.Separate PERIPHERAL BLOOD MONONUCLEAR CELL as mentioned above and extract albumen.In addition, the number in the joint of record C proteins C reactive (CRP) value, tendernesss (tender) and swelling, doctor's the overall evaluation, patient's self evaluation, pain scores and disabled scoring and the ACR that calculates each patient mark, and (ACR is the standard of basis by American College of Rheumatology formulation, calculate based on above-mentioned measurement, be used to estimate the scoring of the effect of drug therapy RA; ACR 20, ACR 50 and ACR 70 represent 20%, 50% and 70% raising of this scoring respectively).
The A that is undertaken by Western blotting (WB)
3The analysis of AR protein expression level
Carry out the Western engram analysis (WB) of synovia, pawl, spleen and lymph node according to following scheme.With freezing PBS rinsing sample and with sample transfer to freezing lysis buffer (TNN damping fluid, 50mM Tris pH of buffer=7.5,150mM NaCl, NP 40).By centrifugal 10 minutes removal cell fragments under 7500xg.Use Bio-Rad protein determination coloring agent to determine protein concentration.The polyacrylamide gel of use 12% separates the sample (50 μ g) of equivalent by SDS-PAGE.Then with the albumen electrotransfection that decomposes to nitrocellulose filter (Schleicher ﹠amp; Schuell, Keene, NH, USA) on.With 1% BSA closing membrane, use the first anti-A then
3The antibody of AR (dilutability 1: 1000) was 4 ℃ of following incubations 24 hours.Wash trace then and at room temperature used the second antibody incubation 1 hour.Use BCIP/NBT colour development kit (Promega, Madison, W1, USA) record band.
The result
A
3AR is raised in inflammation and hematopoietic tissue
Analyze definite A by WB
3The expression of AR in the AIA model.For this reason, obtain also described in material and method, to analyze from Inflamed tissue's (pawl) or from the protein extract of procedure for peripheral blood tissue (peripheral blood lymphocytes, lymph node and spleen).Figure 1A-1D has shown the WB analysis result, and this result also is shown in the histogram of the correspondence that average result and standard deviation are provided.As shown, A
3AR (is raised among Figure 1B-1D) at Inflamed tissue (Figure 1A) and procedure for peripheral blood tissue.
A
3The expression of AR in the AIA model is also relevant with disease clinical score (Fig. 2), and this is inflammation and A
3Association between AR expresses provides further evidence.
CF101 suppresses the development of AIA
After immunity about 21 days, arthritis took place in most of animals through vehicle treated gradually.CF101 handle (10 μ g/kg, every day twice oral administration, after the immunity the 14th day) and methotrexate (MTX) processing cause the remarkable minimizing of disease severity, the result of two kinds of medicines is closely similar, as being estimated by the arthritis clinical score.Disease peaked at the 21st to 28 day and observes the ceiling effect (Fig. 3) of CF101 or MTX in these days.
A
3AR highly expresses in the inflammation tissue of AIA rat and procedure for peripheral blood tissue
In healthy pawl and synovial tissue, detect low A
3The AR expression.In deriving from the inflammation tissue of AIA rat, notice A
3The remarkable increase of AR protein expression level (Fig. 4 A-4B).After IB-MECA handles, A
3The AR level is reduced (Fig. 4 A-4B) at once.In the periphery hematopoietic tissue, notice similar pattern, promptly in the spleen of the animal that derives from not test (N.T.) and lymph node (LN), notice low A
3The AR expression is from the low expression (Fig. 5 A-5C) in the rat tissue of high expression level in the tissue of AIA and IB-MECA processing.In the LN of the AIA rat that the MTX that uses by oneself handles, observe similar A
3AR express spectra (Fig. 6).
In deriving from the MNC of RA, find high A
3AR expresses the low expression among the health volunteer
In from health volunteer's MNC, find low A
3The AR expression, and in deriving from RA patient's MNC, detect high expressed (Fig. 7).
A
3Correlativity between AR expression and the clinical efficacy parameter
Extract blood from 17 RA patients that participate in clinical research, described clinical research detects the influence of CF101 to the performance of the disease among these patients.After the removing phase of previous treatment and extract blood after the treatment at 3 months then, separate PERIPHERAL BLOOD MONONUCLEAR CELL (PBMNC) and determine A in these cells
3The level of AR.As can be seen in Figure 8, there are 5 to be the non-responder among these 17 patients, be these patients even the reaction that does not obtain ACR20, and other 13 patients are respondents, because it has the reaction of ACR20 at least (as seeing in Fig. 8,3 reactions with ACR70 are arranged, 5 reactions that obtain ACR50, other 4 reactions that only obtain ACR20 among the respondent).
As can further seeing in Fig. 8, all ACR50 and ACR70 respondent all have initial high-level A
3AR, after 3 months treatment, this high level reduces, and in the non-responder A
3The AR level does not change basically.By " * " " patient of mark (patient number 1517); although there is not ACR reaction (variation according to CRP level (be used for calculating a parameter of the parameter of ACR scoring-referring to following) is lower than 0% the fact), on other parameters number that particularly touch a tender spot in swelling and joint, have the improvement of highly significant.
These data clearly prove uses A
3The AR level (is particularly utilized A with the prediction patient to the anti-inflammatory medicinal treatment
3The AR activator is as this therapy of disease modification medicine) the ability of reaction.
A in the RA patient of methotrexate for treatment
3The rise of AR expression
Before the therapy of using methotrexate begins and gather blood sample from 4 RA patients afterwards.Separate PBMNC and measure A as mentioned above
3The level of AR.Being shown in result among Fig. 9 proves and uses the therapy of methotrexate to induce A
3The increase of AR level.
The medical history in these data presentation past, the medicinal treatment of particularly passing by can influence the A among the PBMNC
3Therefore the AR level may need to consider this medical history in order to carry out method of the present invention.
Claims (20)
1. determine the method for inflammatory conditions among the experimenter, it comprises, determines from the A in this experimenter's the leucocyte (WBC)
3Adenosine receptor (A
3AR) expression, A
3The high level expression of AR shows the inflammatory conditions among this experimenter.
2. the process of claim 1 wherein the described A among the WBC
3AR expression levels and control level relatively, described control level is the A among health volunteer's the normal WBC
3The expression of AR, or the A of expression normal condition
3The standard reference level that AR expresses.
3. the process of claim 1 wherein that described inflammatory conditions is the result of autoimmune disease.
4. the method for claim 3, wherein said autoimmune disease is rheumatoid arthritis (RA).
5. determine the method for the severity of inflammatory conditions among the experimenter, it comprises the A among the WBC that determines the experimenter
3AR expression and with A in the cell
3The expression of AR and the level of predetermined standard compare, and described predetermined standard makes A
3The severity of AR expression and infection takes place related.
6. the method for claim 5, wherein said inflammatory conditions is the result of autoimmune disease.
7. the method for claim 6, wherein said autoimmune disease is rheumatoid arthritis (RA).
8. be used for determining the method for effect of experimenter's anti-inflammatory treatment processing, this therapy comprises to this experimenter uses A
3The AR activator is included on two or more time points in succession and determines from the A in this experimenter's the leucocyte (WBC)
3The expression of AR, at least one described time point is during anti-inflammatory treatment, and wherein the difference of level is represented the effect of this medicinal treatment.
9. the method for claim 8, wherein gather one or more first samples on the time point before the treatment beginning, and gather one or more second samples on the time point during treating, wherein compare A in one or more second samples with one or more first samples
3It is effective that the AR expression levels reduces this treatment of expression.
10. the method for claim 8, wherein gather one or more first samples on the time point during treating, and gather one or more second samples on the time point during treating after the time point of one or more first samples, wherein compare the A in described one or more second samples with one or more first samples
3The reduction of AR expression represents that this treatment is effective.
11. the method for claim 8, wherein gather one or more samples on the time point during treating, and gather one or more second samples on the time point after described treatment is interrupted, wherein compare the A in one or more second samples with one or more first samples
3The increase of AR expression represents that this treatment is effective.
12. the method for claim 8, wherein said therapeutic treatment comprises anti-inflammatory drug.
13. the method for claim 8, wherein said inflammatory conditions are the results of autoimmune disease.
14. the method for claim 13, wherein said autoimmune disease rheumatoid arthritis (RA).
15. the experimenter who is used to select to suffer certain inflammatory disease is to accept the method for anti-inflammatory treatment processing, described processing comprises to described experimenter uses A
3Adenosine receptor (A
3AR) activator, this method comprise the A in the leucocyte (WBC) of determining this experimenter
3The expression of AR, and if described level be higher than predetermined level then select this experimenter to accept described anti-inflammatory treatment processing.
16. the method for claim 15 is wherein gathered the sample of described WBC from the experimenter before accepting anti-inflammatory therapy.
17. the method for claim 15, wherein said inflammatory conditions are the results of autoimmune disease.
18. the method for claim 18, wherein said autoimmune disease are rheumatoid arthritis (RA).
19. the method for claim 15, wherein said anti-inflammatory treatment processing comprises the IB-MECA that the anti-inflammatory amount is provided to described experimenter.
20. the method for claim 15, this method are used for being chosen in the candidate that the anti-inflammatory treatment processing is accepted in clinical research.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63219804P | 2004-12-02 | 2004-12-02 | |
| US60/632,198 | 2004-12-02 | ||
| US65771805P | 2005-03-03 | 2005-03-03 | |
| US60/657,718 | 2005-03-03 | ||
| PCT/IL2005/001279 WO2006059327A1 (en) | 2004-12-02 | 2005-11-30 | A biological marker for inflammation |
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